The process by which RNA is separated from other molecules, in particular other cellular components such as proteins, carbohydrates, lipids and DNA is widely known and described in the literature. It is a key process in the understanding of gene function and structure and drug development. Furthermore nucleic acid based diagnostic procedures for detecting RNA viruses such as HCV and HIV absolutely require the viral genomic RNA to be isolated in an intact and relatively pure form. In order to separate RNA in an intact and pure form from other biological material it is necessary to rapidly inactivate all ribonucleases that are present and separate the RNA based on a biophysical property unique to the RNA. However, due to the similar physical characteristics of RNA and DNA, RNA preparations are frequently if not always contaminated with DNA, leading to major difficulties in the analysis of results.
Currently mRNA is separated from genomic DNA by using a method based on oligo (dT) interacting with the poly A tail (Aviv and Leder., Proc. Natl. Acad. Sci. USA. 69, 1408–1412 (1972). However, A-rich DNA sequences are co-purified with this method leading to mRNA contaminated with A-rich DNA. Alternatively, nucleases that are specific for either RNA or DNA can be employed. Such highly purified enzymes are costly to use and frequently require removal before the nucleic acid can be used. For example RNase free, DNase must be removed by phenol extraction or heat inactivation otherwise it will destroy for example PCR primers or other DNA based reagents in all post-nuclease reactions. Another method is to use a mixture of phenol, chloroform, isoamyl alcohol (50:49:1); whereby DNA preferentially partitions into the organic phase whilst RNA remains in the aqueous phase. This method at best still leaves a significant amount of DNA contaminating the RNA and hence is of limited practical use. Another method is the TRI REAGENT™ (Molecular Research Centre, Inc) that allows the simultaneous separation of DNA, RNA and proteins. However, it requires careful separation of different phases from each other and subsequent centrifugation steps whilst not assuring complete separation of DNA from the RNA due to the difficulty of pipetting small volumes of liquid without cross-contamination. Another method is anion exchange chromatography which can separate RNA from DNA but the expense and difficulty of setting up the column as well as the restriction to purifying only small nucleic acids precludes its use from the majority of laboratories. Numerous methods to purify RNA are described in general texts (Jones et al., (1994) in RNA Isolation and Analysis. Bios. Oxford., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, CSH.).